Biolarvicidal compound gymnemagenol isolated from leaf extract of miracle fruit plant, Gymnema sylvestre (Retz) Schult against malaria and filariasis vectors

Owing to the fact that the application of synthetic larvicide has envenomed the surroundings as well as non-target organisms, natural products of plant origin with insecticidal properties have been tried as an indigenous method for the control of a variety of insect pests and vectors in the recent past. Insecticides of plant origin have been extensively used on agricultural pests and, to a very limited extent, against insect vectors of public health importance, which deserve careful and thorough screening. The use of plant extracts for insect control has several appealing features as these are generally more biodegradable, less hazardous and a rich storehouse of chemicals of diverse biological activities. Moreover, herbal sources give a lead for discovering new insecticides. Therefore, biologically active plant materials have attracted considerable interest in mosquito control study in recent times. The crude leaf extracts of Gymnema sylvestre (Retz) Schult (Asclepiadaceae) and purified gymnemagenol compound were studied against the early fourth-instar larvae of Anopheles subpictus Grassi and Culex quinquefasciatus Say (Diptera: Culicidae). In the present study, bioassay-guided fractionation of petroleum ether leaf extract of G. sylvestre led to the separation and identification of gymnemagenol as a potential new antiparasitic compound. Phytochemical analysis of G. sylvestre leaves revealed the presence of active constituents such as carbohydrates, saponins, phytosterols, phenols, flavonoids and tannins. However, cardiac glycosides and phlobatannins are absent in the plant extracts. Quantitative analysis results suggested that saponin (5%) was present in a high concentration followed by tannins (1.0%). The 50 g powder was loaded on silica gel column and eluted with chloroform-methanol-water as eluents. From that, 16 mg pure saponin compound was isolated and analysed by thin layer chromatography using chloroform and methanol as the solvent systems. The structure of the purified triterpenoid fraction was established from infrared (IR), ultraviolet (UV), (1)H nuclear magnetic resonance (NMR), (13)C NMR and mass spectral data. The carbon skeleton of the compound was obtained by (13)C NMR spectroscopy. The chemical shift assignments obtained for gymnemagenol from (1)H NMR correspond to the molecular formula C(30)H(50)O(4). The compound was identified as 3β, 16β, 28, 29-tetrahydroxyolean-12-ene (gymnemagenol sapogenin). Parasite larvae were exposed to varying concentrations of purified compound gymnemagenol for 24 h. The results suggested that the larval mortality effects of the compound were 28%, 69%, 100% and 31%, 63%, 100% at 6, 12 and 24 h against A. subpictus and C. quinquefasciatus, respectively. In the present study, the per cent mortality were 100, 86, 67, 36, 21 and 100, 78, 59, 38 and 19 observed in the concentrations of 1,000, 500, 250, 125 and 62.75 ppm against the fourth-instar larvae of A. subpitcus and C. quinquefasciatus, respectively. The purified compound gymnemagenol was tested in concentrations of 80, 40, 20, 10 and 5 ppm, and the per cent mortality were 100, 72, 53, 30 and 15 against A. subpitcus and 100, 89, 61, 42 and 30 against C. quinquefasciatus, respectively. The larvicidal crude leaf extract of G. sylvestre showed the highest mortality in the concentration of 1,000 ppm against the larvae of A. subpictus (LC(50) = 166.28 ppm, r (2) = 0.807) and against the larvae of C. quinquefasciatus (LC(50) = 186.55 ppm, r (2) = 0.884), respectively. The maximum efficacy was observed in gymnemagenol compound with LC(50) and r (2) values against the larvae of A. subpictus (22.99 ppm, 0.922) and against C. quinquefasciatus (15.92 ppm, 0.854), respectively. The control (distilled water) showed nil mortality in the concurrent assay.